Euglycemia can be restored in patients with Type 1 diabetes by pancreatic islet transplants. Problems arising from the shortage of tissue and/or islets must be overcome for this treatment to be widely applied. To address this problem, we are developing renewable cell-based systems for transplant by directing differentiation of human embryonic stem cells or pancreatic progenitor cells into insulin producing beta cells, or by activating existing beta cell populations to proliferate without loss of insulin production. We apply proteomics-based technologies and biochemical methods to study the relationship between protein expression patterns and signal transduction. Protein expression profiles of human islets using two-dimensional (2D) electrophoresis and mass spectrometry (MS) found large changes in protein expression linked to phosphatidylinositol 3-OH kinase (PI 3-kinase), an activator of multiple signaling pathways, including proliferation and cell survival. We hypothesize that changes in protein expression that occur during islet differentiation create new protein:protein interactions that alter signals processed through the PI 3-kinase pathway. In the proposed studies, we will use using 2D electrophoresis and MS to identify proteins in endocrine progenitor cells, mature beta cells, and expanded beta cells whose sub-cellular distribution and expression profiles change upon activation or inhibition of PI 3-kinase and compiled into a central database. The data generated will be used to identify proteins that regulate the activity and localization of PDK-1 (3- phosphoinositide dependent kinase-1), a protein kinase directly downstream of PI 3-kinase that regulates cell survival and proliferation. Directed proteomics studies in our laboratory indicate at least 30 PDK-1 binding proteins exist. We will identify these proteins and search for changes in their ability to bind PDK-1 as fetal islets differentiate into mature beta cells. Data generated will lead to a greater understanding of how PI 3-kinase regulates beta cell development and proliferation.